Overcoming donor variability and risks associated with fecal microbiota transplants through bacteriophage-mediated treatments.

BACKGROUND: Fecal microbiota transplantation (FMT) and fecal virome transplantation (FVT, sterile filtrated donor feces) have been effective in treating recurrent Clostridioides difficile infections, possibly through bacteriophage-mediated modulation of the gut microbiome. However, challenges like donor variability, costly screening, coupled with concerns over pathogen transfer (incl. eukaryotic viruses) with FMT or FVT hinder their wider clinical application in treating less acute diseases. METHODS: To overcome these challenges, we developed methods to broaden FVT's clinical application while maintaining efficacy and increasing safety. Specifically, we employed the following approaches: (1) chemostat-fermentation to reproduce the bacteriophage FVT donor component and remove eukaryotic viruses (FVT-ChP), (2) solvent-detergent treatment to inactivate enveloped viruses (FVT-SDT), and (3) pyronin-Y treatment to inhibit RNA virus replication (FVT-PyT). We assessed the efficacy of these processed FVTs in a C. difficile infection mouse model and compared them with untreated FVT (FVT-UnT), FMT, and saline. RESULTS: FVT-SDT, FVT-UnT, and FVT-ChP reduced the incidence of mice reaching the humane endpoint (0/8, 2/7, and 3/8, respectively) compared to FMT, FVT-PyT, and saline (5/8, 7/8, and 5/7, respectively) and significantly reduced the load of colonizing C. difficile cells and associated toxin A/B levels. There was a potential elimination of C. difficile colonization, with seven out of eight mice treated with FVT-SDT testing negative with qPCR. In contrast, all other treatments exhibited the continued presence of C. difficile. Moreover, the results were supported by changes in the gut microbiome profiles, cecal cytokine levels, and histopathological findings. Assessment of viral engraftment following FMT/FVT treatment and host-phage correlations analysis suggested that transfer of phages likely were an important contributing factor associated with treatment efficacy. CONCLUSIONS: This proof-of-concept study shows that specific modifications of FVT hold promise in addressing challenges related to donor variability and infection risks. Two strategies lead to treatments significantly limiting C. difficile colonization in mice, with solvent/detergent treatment and chemostat propagation of donor phages emerging as promising approaches. Video Abstract.

Metaproteomics reveals parallel utilization of colonic mucin glycans and dietary fibers by the human gut microbiota.

A diet lacking dietary fibers promotes the expansion of gut microbiota members that can degrade host glycans, such as those on mucins. The microbial foraging on mucin has been associated with disruptions of the gut-protective mucus layer and colonic inflammation. Yet, it remains unclear how the co-utilization of mucin and dietary fibers affects the microbiota composition and metabolic activity. Here, we used 14 dietary fibers and porcine colonic and gastric mucins to study the dynamics of mucin and dietary fiber utilization by the human fecal microbiota in vitro. Combining metaproteome and metabolites analyses revealed the central role of the Bacteroides genus in the utilization of complex fibers together with mucin while Akkermansia muciniphila was the main utilizer of sole porcine colonic mucin but not gastric mucin. This study gives a broad overview of the colonic environment in response to dietary and host glycan availability.

Transfer of modified gut viromes improves symptoms associated with metabolic syndrome in obese male mice.

Metabolic syndrome encompasses amongst other conditions like obesity and type-2 diabetes and is associated with gut microbiome (GM) dysbiosis. Fecal microbiota transplantation (FMT) has been explored to treat metabolic syndrome by restoring the GM; however, concerns on accidentally transferring pathogenic microbes remain. As a safer alternative, fecal virome transplantation (FVT, sterile-filtrated feces) has the advantage over FMT in that mainly bacteriophages are transferred. FVT from lean male donors have shown promise in alleviating the metabolic effects of high-fat diet in a preclinical mouse study. However, FVT still carries the risk of eukaryotic viral infections. To address this, recently developed methods are applied for removing or inactivating eukaryotic viruses in the viral component of FVT. Modified FVTs are compared with unmodified FVT and saline in a diet-induced obesity model on male C57BL/6 N mice. Contrasted with obese control, mice administered a modified FVT (nearly depleted for eukaryotic viruses) exhibits enhanced blood glucose clearance but not weight loss. The unmodified FVT improves liver pathology and reduces the proportions of immune cells in the adipose tissue with a non-uniform response. GM analysis suggests that bacteriophage-mediated GM modulation influences outcomes. Optimizing these approaches could lead to the development of safe bacteriophage-based therapies targeting metabolic syndrome through GM restoration.

Prevotella enterotype associates with diets supporting acidic faecal pH and production of propionic acid by microbiota.

Metabolism of dietary fibres by colon microbiota plays an important role for human health. Personal data from a nutrition study (57 subjects) were analysed to elucidate quantitative associations between the diet, faecal microbiome, organic acid concentrations and pH. Ratios of the predominant acids acetate, butyrate and propionate ranged from 1:0.67:0.27 to 1:0.17:0.36. Pectin-rich diets resulted in higher faecal acetate concentrations. Negative correlation between faecal pH and BSS was observed. Higher faecal pH and lower acid concentrations were related to the higher abundance of amino acid degrading Clostridium, Odoribacter and Eubacterium coprostanoligenes, which are weak carbohydrate fermenting taxa. Propionic acid correlated especially to high abundance of Prevotella and low abundance of proteobacteria. The acetate to propionate ratio of the Prevotella enterotype was about half of that of the Bacteroides enterotype. Based on the results we suggest the measurement of faecal pH and organic acid composition for research and diagnostic purposes.

Acidic pH enhances butyrate production from pectin by faecal microbiota.

Environmental pH and gut transit rate are the key factors determining the dynamics of colonic microbiota. In this study, the effect of changing pH on the composition and metabolism of pooled faecal microbiota was elucidated at physiologically relevant dilution rates Dhigh = 0.2 and Dlow = 0.05 1/h. The results showed the best adaptability of Bacteroides ovatus within the pH range 6.0-8.0 at both dilution rates. The butyrate producing Faecalibacterium and Coprococcus comes were extremely sensitive to pH > 7.5, while the abundance of Akkermansia muciniphila increased significantly at pH >7 at Dhigh, causing a pH-dependant shift in the dynamics of mucin degrading species. Increased gas formation was observed at pH < 6.5. Substantially more CO2 was produced at Dlow than at Dhigh (18-29 vs 12-23 mmol per L medium, respectively). Methane was produced only at Dlow and pH > 7, consistent with the simultaneous increased abundance of Methanobrevibacter smithii. Our study confirmed the importance of pH in the development of faecal microbiota in pectin-supplemented medium. Fermentation of other dietary fibres can be studied using the same approach. The significance of pH should be more emphasized in gut research and diagnostics.

Microbiome of root vegetables-a source of gluten-degrading bacteria.

Gluten is a cereal protein that is incompletely digested by human proteolytic enzymes that create immunogenic peptides that accumulate in the gastrointestinal tract (GIT). Although both environmental and human bacteria have been shown to expedite gluten hydrolysis, gluten intolerance is a growing concern. Here we hypothesize that together with food, we acquire environmental bacteria that could impact our GIT with gluten-degrading bacteria. Using in vitro gastrointestinal simulation conditions, we evaluated the capacity of endophytic bacteria that inhabit root vegetables, potato (Solanum tuberosum), carrot (Daucus sativus), beet (Beta vulgaris), and topinambur (Jerusalem artichoke) (Helianthus tuberosus), to resist these conditions and degrade gluten. By 16S rDNA sequencing, we discovered that bacteria from the families Enterobacteriaceae, Bacillaceae, and Clostridiaceae most effectively multiply in conditions similar to the human GIT (microoxic conditions, 37 °C) while utilizing vegetable material and gluten as nutrients. Additionally, we used stomach simulation (1 h, pH 3) and intestinal simulation (1 h, bile salts 0.4%) treatments. The bacteria that survived this treatment retained the ability to degrade gluten epitopes but at lower levels. Four bacterial strains belonging to species Bacillus pumilus, Clostridium subterminale, and Clostridium sporogenes isolated from vegetable roots produced proteases with postproline cleaving activity that successfully neutralized the toxic immunogenic epitopes. KEY POINTS: • Bacteria from root vegetables can degrade gluten. • Some of these bacteria can resist conditions mimicking gastrointestinal tract.

Use of Changestat for Growth Rate Studies of Gut Microbiota.

Human colon microbiota, composed of hundreds of different species, is closely associated with several health conditions. Controlled in vitro cultivation and up-to-date analytical methods make possible the systematic evaluation of the underlying mechanisms of complex interactions between the members of microbial consortia. Information on reproducing fecal microbial consortia can be used for various clinical and biotechnological applications. In this study, chemostat and changestat cultures were used to elucidate the effects of the physiologically relevant range of dilution rates on the growth and metabolism of adult fecal microbiota. The dilution rate was kept either at D = 0.05 or D = 0.2 1/h in chemostat cultures, while gradually changing from 0.05 to 0.2 1/h in the A-stat and from 0.2 to 0.05 1/h in the De-stat. Apple pectin as a substrate was used in the chemostat experiments and apple pectin or birch xylan in the changestat experiments, in the presence of porcine mucin in all cases. The analyses were comprised of HPLC for organic acids, UPLC for amino acids, GC for gas composition, 16S-rDNA sequencing for microbial composition, and growth parameter calculations. It was shown that the abundance of most bacterial taxa was determined by the dilution rate on both substrates. Bacteroides ovatus, Bacteroides vulgatus, and Faecalibacterium were prevalent within the whole range of dilution rates. Akkermansia muciniphila and Ruminococcaceae UCG-013 were significantly enriched at D = 0.05 1/h, while Bacteroides caccae, Lachnospiraceae unclassified and Escherichia coli clearly preferred D = 0.2 1/h. In the chemostat cultures, the production of organic acids and gases from pectin was related to the dilution rate. The ratio of acetate, propionate and butyrate was 5:2:1 (D = 0.05 1/h) and 14:2:1 (D = 0.2 1/h). It was shown that the growth rate-related characteristics of the fecal microbiota were concise in both directions between D = 0.05 and 0.2 1/h. Reproducible adaptation of the fecal microbiota was shown in the continuous culture with a changing dilution rate: changestat. Consortia cultivation is a promising approach for research purposes and several biotechnological applications, including the production of multi-strain probiotics and fecal transplantation mixtures.

The composition of faecal microbiota is related to the amount and variety of dietary fibres.

For normal gut and body function, the diet should contain variety of dietary fibres. To elucidate the links between food intake, especially the variety of dietary fibres, faecal microbiota, body mass index and content of blood lipids, 59 healthy subjects on common Estonian diet were enrolled. The dietary records were analysed at nutrient level while seven categories of fibres were characterised to differentiate variety of fibre profiles consumed. The data of the high fibre (HF) intake (>15.1 g/1000 kcal) and the low fibre (LF) intake (<9.4 g/1000 kcal) groups were comparatively evaluated. LF diets associated with Collinsella, Coprococcus and Dorea, and higher consumption of meat and white flour products while HF diet with Roseburia, Bacteroides xylanisolvens and Oxalobacter formigenes, and arabinoxylan and pectin rich cereals and vegetables. Based on the results, modulation of the colon microbiota can be suggested by careful variation and enrichment of dietary fibre sources.

Composition and metabolism of fecal microbiota from normal and overweight children are differentially affected by melibiose, raffinose and raffinose-derived fructans.

The aim of the study was to investigate the metabolism of non-digestible oligo- and polysaccharides by fecal microbiota, using isothermal microcalorimetry. The five tested substrates were raffinose, melibiose, a mixture of oligo- and polysaccharides produced from raffinose by levansucrase, levan synthesized from raffinose, and levan from timothy grass. Two inocula were comprised of pooled fecal samples from overweight or normal-weight children, from healthy adult volunteers and a pure culture of Bacteroides thetaiotaomicron as a reference bacterium for colon microbiota. The growth was analyzed based on the heat evolution curves, and the production of organic acids and gases. Taxonomic profiles of the microbiota were assessed by 16S rDNA sequencing. Raffinose and melibiose promoted the growth of bifidobacteria in all fecal pools. Several pool-specific substrate-related responses to raffinose and melibiose were revealed. Lactate-producing bacteria (Streptococcus and Enterococcus) became enriched in the pool of overweight children resulting in lactic acid as the major fermentation product on short saccharides. Acetic and butyric acids were prevalent at fermentation in the normal-weight pool coinciding with the enrichment of Catenibacterium. In the adult pool, the specific promotion of Bacteroides and Lachnospiraceae by levans was disclosed. In the fecal pool of normal-weight children, levans stimulated the growth of Senegalimassilia and Lachnoclostridium and this particular pool also showed the highest maximum heat production rate at levan fermentation. Levans and raffinose-derived oligosaccharides, but not raffinose and melibiose were completely fermented by a pure culture of Bacteroides thetaiotaomicron. The main conclusion from the study is that fecal microbiota of normal and overweight children have different compositions and they respond in specific manners to non-digestible oligo- and polysaccharides: raffinose, melibiose, raffinose-derived oligosaccharides and levans. The potential of the tested saccharides to support a healthy balance of colon microbiota requires further studies.

Influence of gemcitabine chemotherapy on the microbiota of pancreatic cancer xenografted mice.

BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) represents the fourth cause of cancer-related death. We aimed to evaluate whether gemcitabine treatment shapes the gut microbiota in a model of PDAC xenografted mice. MATERIALS AND METHODS: Pancreatic cancer xenograft mice were subjected to gemcitabine injection once per week for 3 weeks to assess the tumor volume as compared to control mice injected with normal saline solution. The composition of fecal microbiota, the activation of NF-kB pathway in cancer tissues and the serum metabolomics were further analyzed. RESULTS: Gemcitabine considerably decreases the proportion of Gram- positive Firmicutes (from about 39 to 17%) and the Gram- negative Bacteroidetes (from 38 to 17%) which are the two dominant phyla in the gut of tumor-bearing control mice. This downshift was replaced by an increase of Proteobacteria (Escherichia coli and Aeromonas hydrophila) from 15 up to 32% and Verrucomicrobia (Akkermansia muciniphila) from 5 to 33% in the gut of drug-receiving mice. An overall increase in inflammation-associated bacteria was observed upon gemcitabine. Consistently, activation of the NF-kB canonical pathway was found in cancer tissues from gemcitabine-treated mice. Serum metabolomics revealed a significant decrease of the purine compounds inosine and xanthine, and a decreasing trend for their metabolically-related molecule hypoxanthine. DISCUSSION: Understanding chemotherapy side effects may explain the lack of activity or the chemoresistant processes and it may help to set up strategies to improve the effectiveness of therapy.

Engineered Resistant-Starch (ERS) Diet Shapes Colon Microbiota Profile in Parallel with the Retardation of Tumor Growth in In Vitro and In Vivo Pancreatic Cancer Models.

BACKGROUND/AIMS: Pancreatic cancer (PC) is ranked as the fourth leading cause of cancer-related deaths worldwide. Despite recent advances in treatment options, a modest impact on the outcome of the disease is observed so far. We have previously demonstrated that short-term fasting cycles have the potential to improve the efficacy of chemotherapy against PC. The aim of this study was to assess the effect of an engineered resistant-starch (ERS) mimicking diet on the growth of cancer cell lines in vitro, on the composition of fecal microbiota, and on tumor growth in an in vivo pancreatic cancer mouse xenograft model. MATERIALS AND METHODS: BxPC-3, MIA PaCa-2 and PANC-1 cells were cultured in the control, and in the ERS-mimicking diet culturing condition, to evaluate tumor growth and proliferation pathways. Pancreatic cancer xenograft mice were subjected to an ERS diet to assess tumor volume and weight as compared to mice fed with a control diet. The composition and activity of fecal microbiota were further analyzed in growth experiments by isothermal microcalorimetry. RESULTS: Pancreatic cancer cells cultured in an ERS diet-mimicking medium showed decreased levels of phospho-ERK1/2 (extracellular signal-regulated kinase proteins) and phospho-mTOR (mammalian target of rapamycin) levels, as compared to those cultured in standard medium. Consistently, xenograft pancreatic cancer mice subjected to an ERS diet displayed significant retardation in tumor growth. In in vitro growth experiments, the fecal microbial cultures from mice fed with an ERS diet showed enhanced growth on residual substrates, higher production of formate and lactate, and decreased amounts of propionate, compared to fecal microbiota from mice fed with the control diet. CONCLUSION: A positive effect of the ERS diet on composition and metabolism of mouse fecal microbiota shown in vitro is associated with the decrease of tumor progression in the in vivo PC xenograft mouse model. These results suggest that engineered dietary interventions could be supportive as a synergistic approach to enhance the efficacy of existing cancer treatments in pancreatic cancer patients.

Bacterial polysaccharide levan as stabilizing, non-toxic and functional coating material for microelement-nanoparticles.

Levan, fructose-composed biopolymer of bacterial origin, has potential in biotechnology due to its prebiotic and immunostimulatory properties. In this study levan synthesized by levansucrase from Pseudomonas syringae was thoroughly characterized and used as multifunctional biocompatible coating material for microelement-nanoparticles (NPs) of selenium, iron and cobalt. Transmission electron microscopy (TEM), hydrodynamic size measurements (DLS) and X-ray photoelectron spectroscopy (XPS) showed the interaction of levan with NPs. Levan stabilized the dispersions of NPs, decreased their toxicity and had protective effect on human intestinal cells Caco-2. In addition, levan attached to cobalt NPs remained accessible as a substrate for the colon bacteria Bacteroides thetaiotaomicron. We suggest that the combination of levan and nutritionally important microelements in the form of NPs serves as a first step towards a novel "2 in 1" approach for food supplements to provide safe and efficient delivery of microelements for humans and support beneficial gut microbiota with nutritional oligosaccharides.

Levan Enhances Associated Growth of Bacteroides, Escherichia, Streptococcus and Faecalibacterium in Fecal Microbiota.

The role of dietary fiber in supporting healthy gut microbiota and overall well-being of the host has been revealed in several studies. Here, we show the effect of a bacterial polyfructan levan on the growth dynamics and metabolism of fecal microbiota in vitro by using isothermal microcalorimetry. Eleven fecal samples from healthy donors were incubated in phosphate-buffered defined medium with or without levan supplementation and varying presence of amino acids. The generation of heat, changes in pH and microbiota composition, concentrations of produced and consumed metabolites during the growth were determined. The composition of fecal microbiota and profile of metabolites changed in response to substrate (levan and amino acids) availability. The main products of levan metabolism were acetic, lactic, butyric, propionic and succinic acids and carbon dioxide. Associated growth of levan-degrading (e.g. Bacteroides) and butyric acid-producing (e.g. Faecalibacterium) taxa was observed in levan-supplemented media. The study shows that the capacity of levan and possibly also other dietary fibers/prebiotics to modulate the composition and function of colon microbiota can be predicted by using isothermal microcalorimetry of fecal samples linked to metabolite and consortia analyses.

Survival and synergistic growth of mixed cultures of bifidobacteria and lactobacilli combined with prebiotic oligosaccharides in a gastrointestinal tract simulator.

BACKGROUND: Probiotics, especially in combination with non-digestible oligosaccharides, may balance the gut microflora while multistrain preparations may express an improved functionality over single strain cultures. In vitro gastrointestinal models enable to test survival and growth dynamics of mixed strain probiotics in a controlled, replicable manner. METHODS: The robustness and compatibility of multistrain probiotics composed of bifidobacteria and lactobacilli combined with mixed prebiotics (galacto-, fructo- and xylo-oligosaccharides or galactooligosaccharides and soluble starch) were studied using a dynamic gastrointestinal tract simulator (GITS). The exposure to acid and bile of the upper gastrointestinal tract was followed by dilution with a continuous decrease of the dilution rate (de-celerostat) to simulate the descending nutrient availability of the large intestine. The bacterial numbers and metabolic products were analyzed and the growth parameters determined. RESULTS: The most acid- and bile-resistant strains were Lactobacillus plantarum F44 and L. paracasei F8. Bifidobacterium breve 46 had the highest specific growth rate and, although sensitive to bile exposure, recovered during the dilution phase in most experiments. B. breve 46, L. plantarum F44, and L. paracasei F8 were selected as the most promising strains for further studies. CONCLUSIONS: De-celerostat cultivation can be applied to study the mixed bacterial cultures under defined conditions of decreasing nutrient availability to select a compatible set of strains.

Degradation of Fructans and Production of Propionic Acid by Bacteroides thetaiotaomicron are Enhanced by the Shortage of Amino Acids.

Bacteroides thetaiotaomicron is commonly found in the human colon and stabilizes its ecosystem by catabolism of various polysaccharides. A model of cross-talk between the metabolism of amino acids and fructans in B. thetaiotaomicron was proposed. The growth of B. thetaiotaomicron DSM 2079 in two defined media containing mineral salts and vitamins, and supplemented with either 20 or 2 amino acids, was studied in an isothermal microcalorimeter. The polyfructans inulin (from chicory) and levan (synthesized using levansucrase from Pseudomonas syringae), two fructooligosaccharide preparations with different composition, sucrose and fructose were tested as substrates. The calorimetric power-time curves were substrate specific and typically multiauxic. A surplus of amino acids reduced the consumption of longer oligosaccharides (degree of polymerization > 3). Bacterial growth was not detected either in the carbohydrate free medium containing amino acids or in the medium with inulin as a sole carbohydrate. In amino acid-restricted medium, fermentation leading to acetic acid formation was dominant at the beginning of growth (up to 24 h), followed by increased lactic acid production, and mainly propionic and succinic acids were produced at the end of fermentation. In the medium supplemented with 20 amino acids, the highest production of d-lactate (82 ± 33 mmol/gDW) occurred in parallel with extensive consumption (up to 17 mmol/gDW) of amino acids, especially Ser, Thr, and Asp. The production of Ala and Glu was observed at growth on all substrates, and the production was enhanced under amino acid deficiency. The study revealed the influence of amino acids on fructan metabolism in B. thetaiotaomicron and showed that defined growth media are invaluable in elucidating quantitative metabolic profiles of the bacteria. Levan was shown to act as an easily degradable substrate for B. thetaiotaomicron. The effect of levan on balancing or modifying colon microbiota will be studied in further experiments.

Effect of stress pretreatment on survival of probiotic bacteria in gastrointestinal tract simulator.

The effect of stress pretreatment on survival of probiotic Lactobacillus acidophilus La-5, Lactobacillus rhamnosus GG, and Lactobacillus fermentum ME-3 cultures was investigated in the single bioreactor gastrointestinal tract simulator (GITS). The cultures were pregrown in pH-auxostat, subjected to temperature, acid, or bile stress treatment, fast frozen in liquid nitrogen (LN(2)), and tested for survival in GITS. After LN(2) freezing the colony forming ability of L. rhamnosus GG and L. fermentum ME-3 nonstressed and stressed cells was well retained (average survival of 75.4 +/- 18.3% and 88.0 +/- 7.2%, respectively). L. acidophilus La-5 strain showed good survival of auxostat nonstressed cells after fast freezing (94.2 +/- 15.0), however the survival of stress pretreated cells was considerably lower (30.8 +/- 8.5%). All LN(2) frozen auxostat cultures survived well in the acid phase of the GIT simulation (survival 81 +/- 21%); however, after the bile phase, the colony formation ability of L. acidophilus La-5, L. rhamnosus GG, and L. fermentum ME-3 decreased by approximately 1.4 +/- 0.2, 3.8 +/- 0.3, and 3.5 +/- 1.2 logarithmic units, respectively. No statistically relevant positive effect of stress pretreatments on survival of LN(2) frozen L. acidophilus La-5, L. rhamnosus GG, and L. fermentum ME-3 in GITS was observed.